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Research Summary

We study the molecular mechanisms used by viruses in the family Parvoviridae to interact with, and overwhelm, their mammalian host cells. I focus particularly on the strategies used to uncoat, replicate and package their DNA, and on how they manipulate their nuclear environment. These viruses are common and effective pathogens, and so are important objects of study in their own right, but they also teach us much about the biology of the mammalian cells they infect.

Extensive Research Description

Parvoviruses are among the smallest known viruses, encoding just two genes in a single 5-kilobase DNA chromosome, delivered in a non-enveloped protein capsid 280Å in diameter. These viruses are unique because their genomes are both linear and single-stranded, which has a profound impact on their life cycle. First, because single-stranded DNA is more flexible than duplex DNA, their virions can be remarkably small and dense, which allows them to be transported into the cell nucleus intact, potentially sequestering the latent genome from untimely recognition by host DNA damage circuits. Second, viral gene expression only becomes activated when the cell enters S-phase under normal cell cycle control and provides the virus with a complementary, positive-sense, DNA strand. Thus the virus remains silent, and presumably undetected, in its host cell until that cell elects to enter S-phase. This year our research has focused on two main areas, detailed below. The capsids of a murine parvovirus, MVM, do not disassemble during cell entry, but undergo a series of structural transitions that lead first to exposure of the VP1 N-termini, followed by the triggered expulsion of viral DNA, in a critical 3’-to-5’ orientation.A series of programmed structural shifts occur in the rugged virion shell as it progresses through the entry compartments of its host cell, moving toward the cell nucleus where it will ultimately be replicated. These subtle transitions result in sequential exposure of enzymatic domains and signal motifs that allow the virion to gain access first to the cytoplasm, using a phospholipase A2 module deployed on cryptic VP1-N-terminal peptides to penetrate the cell’s lipid bilayer, and then to be trafficked through the cytoplasm, into the nucleus, and ultimately make its genome accessible to the replication machinery of the cell in a way that both protects it’s genome from modification by damage repair mechanisms and fails to elicit potentially-inactivating cell cycle checkpoints. Exciting developments in this work focus on the discovery of a previously-unsuspected divalent cation-modulated uncoating reaction that ejects the DNA, in a 3’-to-5’ direction, from the otherwise intact virus particle. This structural rearrangement thus provides a potential mechanism for uncoating DNA that could be specifically triggered in the cell nucleus at the start of S-phase, allowing the capsid to sequester the genome from nuclear circuits until that time. Moreover, the reaction optimally presents the primer-template end of the genome to the cellular transcription and DNA replication machinery. In vitro, cation-depleted virions remain stable at 4oC, and only uncoat when the temperature is raised to 37oC. This results in release of most of the viral genome but leaves the extreme 5’ end still tightly associated with the intact capsid. Uncoating is not seen in particles with subgenomic DNA, suggesting that pressure exerted by the full length genome influences this rearrangement. Uncoating can be effectively prevented by addition of 1mM CaCl2 or MgCl2 prior to incubation at 37oC, suggesting that a readily accessible cation binding site on the particle modulates the release mechanism. In vitro, the uncoated genomes support complementary strand synthesis by various DNA polymerases, but the resulting duplex products remain firmly attached to the capsid via their right-hand end. If a similar uncoating mechanism were to operate in vivo, the first duplex viral DNAs in the cell, which function as the first viral transcription templates, would be physically linked to an intact capsid, which could influence their position in the nucleus and/or their ability to interact with specific host transcription complexes.

Viral chromatin and the establishment of a specialized viral replication compartment.With the onset of S-phase MVM infected cells embark on normal cellular DNA synthesis, but within two hours this synthesis is disrupted, and there follows a lag period of 3-4 hours, during which time viral proteins accumulate and effectively reprogram the nuclear environment to favor viral amplification. While largely mediated by host cell machinery, viral DNA replication breaks with the convention of coordinated bidirectional leading and lagging strand DNA synthesis that is so carefully policed in mammalian cells. Thus, it creates a series of DNA structures that appear to elicit strong damage responses, and while our research efforts previously focused on molecular details of the virus that allow its efficient replication, we are now turning our attention to how the virus may invoke, utilize and/or inactivate these cellular damage responses. Part of its avoidance strategy appears to involve the elaboration of a unique form of chromatin, which ChIP analysis suggests incorporates both cellular histones and many copies of NS1, the major viral non-structural protein. This is organized by NS1 oligomers binding site-specifically to variable clusters of individual low-affinity sequence blocks that are distributed throughout the viral genome. We have also identified two major types of damage response that become activated during viral amplification: hyper-phosphorylation of the 32kDa subunit of replication protein A (RPA), which typically biases the synthetic machinery of the cell to support repair rather than replicative synthesis; and phosphorylation of the C-terminal tail of histone variant H2AX, followed by the apparent translocation and sequestration of gH2AX/MDC1 repair foci in the outer regions of viral replication centers. We are currently exploring the role and fate of these proteins, and their downstream effectors, asking whether the virus co-opts them for its own use and/or induces their inactivation, in order to abrogate negative aspects of double-strand break repair.

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